Chronic Tyrosinemia Associated with 4-Hydroxyphenylpyruvate Dioxygenase Deficiency with Acute Intermittent Ataxia and Without Visceral and Bone Involvement

Home , Dioxygenase, Hawkinsinuria, Transamination, Tyrosinemia type II

Pediatr. Res. 17: 25-29 (1983)

Chronic Tyrosinemia Associated with 4-Hydroxyphenylpyruvate Dioxygenase Deficiency with Acute Intermittent Ataxia and without Visceral and Bone Involvement

O. GIARDINI, A. CANTANI, N.G. KENNAWAy(28) AND P. D'EUFEMIA r Clinica Pediatrica dell'Universita, Roma, Italy [0. G., A.C., P. D'E.j and Department ofMedical Genetics, Oregon Health Sciences University, Portland, Oregon USA [N.G.K.j

Summary drolase (EC 3.7.1.2) has been demonstrated in several patients (18) and is now felt to be the primary enzyme defect in this A 17-month-old girl, with acute intermittent ataxia and drowsi disease. ness, had hypertyrosinemia (serum tyrosine, 62 JLmole/dl) and Tyrosinemia type II has been described in over twenty patients, phenolic aciduria in the absence of hepatic, renal, eye or skin most ofwhom have manifest the Richner-Hanhart syndrome with

lesions. Serum methionine and urinary acid con severe persistent keratitis and more variable hyperkeratosis on the centrations were normal. Her psychomotor development was also fmgers and palms ofthe hands and soles ofthe feet. These lesions normal. Protein restriction and vitamin C therapy failed to correct respond completely to dietary tyrosine restriction. Blood tyrosine the biochemical abnormality. Liver biopsy was histologically nor is greatly elevated and there is massive tyrosyluria. Absent activity mal. ofhepatic cytosol TAT has been demonstrated in two patients (9, Analysisof the enzymes in the liver biopsy, taken at 25 months 16) and a milder deficiency has been reported in two other of age, showed no detectable activity of 4-hydroxyphenylpyruvate patients, in liver (Il) and fibroblasts (4) respectively. dioxygenase (4HPPD), either in whole homogenate or cytosol Another form oftyrosinemia has been reported in two childrep. fraction. Mixing experiments revealed no inhibitor of either with severe metabolic acidosis who excreted the unusual tyrosine 4HPPD or tyrosine aminotransferase (TAT). metabolites hawkinsin and cis- and trans-4-hydroxycyclohexyla TAT in unfractionated liver was 0.23 JLmole/mg protein/h (con cetic acids in the urine (3, 22, 26). The' primary abnormality is trol, 0.10-0.30 JLmole/mg protein/h; n = 5). In mitochondria, TAT thought to be in the rearrangement of an intermediate formed in was 0.24 JLmole/mg protein/h (control, 0.09-0.12 JLmole/mg pro the 4HPPD reaction. tein/h; n = 3) whereas in cytosol fraction it was 0.23 JLmole/mg We now report a case of persistent tyrosinemia and tyrosyluria protein/h (control, 0.27-0.44 JLmole/mg protein/h; n = 3). Gluta associated with deficient activity of 4HPPD in liver. The clinical mate dehydrogenase activity appeared in the cytosol fraction picture was characterized by sporadic ataxia and drowsiness in a suggesting some rupture of mitochondria during fractionation of child who was otherwise normal. the patient's liver and indicating that true cytosol TAT might be somewhat lower than indicated; however, the kinetics of the pa tient's cytosol TAT were normal: Km for tyrosine, 4.5 X 10-3 M MATERIALS AND METHODS (control, 4.0 X 10-3 M); Km for a-ketoglutarate, 98 X 10-6 M 6 Case report. (control,75 X 10- M); approximate Km for pyridoxal phosphate, K.W. was the second child of healthy, unrelated, 6 6 Egyptian parents. Pregnancy and delivery were uncomplicated. 2.1 X 10- M (control, 4.0 X 10- M). Vmax in patient liver was 0.37 JLmole/mg protein/h (control, 0.88 JLmole/mg protein/h). Birthweight was 3200 g and the neonatal period was normal. No These data argue against a primary abnormality of TAT but are problems occurred until 17 months of age, when the child was consistent with a defect of 4HPPD; thus, this patient appears to admitted to the Pediatric Clinic of Rome University because of represent a previously undescribed form of tyrosinemia. acute ataxia followed by drowsiness. On examination she dem onstrated confusion, motor incoordination, muscular hypotonia and absent tendon reflexes. The pupils were unreactive to light, Speculation there was no response to painful stimuli, breathing was noisy and Demonstration of deficient activity of 4-hydroxyphenylpyruvate the liver edge was 2 em below the costal margin. The examination dioxygenase in a patient with a unique form of tyrosinemia raises was otherwise normal. Blood glucose was normal. Routine toxi the possibility that a similar defect may be present in several other cologic analysis ofserum and urine by gas-liquid chromatography patients with atypical forms of tyrosinemia. revealed no poisonous agents. The child was treated intravenously with fluids, glucose, and electrolytes. After 8 h, response to stimuli and reflexes reappeared There are two well recognized forms of hereditary tyrosinemia. and in the following days, voluntary movements improved al- In the first (type I), associated with failure to thrive, rickets, though her gait was ataxic until day 10. Fanconi syndrome, and progressive liver failure, the levels of During this time, routine laboratory tests, including SGOT (20- blood tyrosine and frequently methionine are elevated and there 38 IU/liter), SGPT (Il-20 IU/liter), total serum protein (8 g/ is increased urinary excretion of tyrosine and its metabolites liter), alkaline phosphatase (110 IU/liter), total serum cholesterol (tyrosyluria) as well as increased o-aminolevulinic acid. Deficient (125 mg/dl), and triglycerides (48 mg/dl) were normal. Glomer- activity of a number of enzymes in the liver, including cytosol ular filtration rate (GFR), creatinine, creatinine clearance, and tyrosine aminotransferase (TAT; EC 2.6.1.5.) and 4-hydroxyphen- phosphate reabsorption were also normal. Plasma amino acids ylpyruvate dioxygenase (4HPPD; EC 1.13.11.27) has been re- (Table I) established the diagnosis of tyrosinemia. Fundus ex- ported. Recently, a deficiency of hepatic fumarylacetoacetate hy- amination, E.E.G., E.C.G., skull x-rays and psychomotor devel- 25 26 GIARDINI ET AL.

Table 1. Serum aminoacid levels (p,mole/dl) Patient Mother Normals' Age 17 months 19 months 27 months 42 months 9-24 months Dietary protein (g/kg/day) normal 1.0 1.0 normal normal normal

Tyrosine 62.4 80.5 49.6 69.6 6.7 1.1-12.2 Phenylalanine 7.6 7.9 6.2 10.2 5.3 2.3-6.9 Methionine 2.6 5.4 3.3 2.8 3.4 0.3-2.9

I From Soupart (24). opment were also normal (LQ. 123). Skeletal x-rays showed mild 1000 X g for 10 min and the supernatant sonicated as described. osteoporosis but no rickets. All fractions were stored at -70°C. After the patient had recovered, an oral loading test with L TAT was measured by the radiochemical procedure ofFellman tyrosine (100 mg/kg in fruit juice acidified with ascorbic acid) was et al. (8, 9) except that the final concentration of a-ketoglutarate performed. The serum tyrosine increased from 80 p,mole/dl at was 10 mM, the buffer was 0.2 M Tris, pH 7.5 and N, N' zero time to 101 p,mole/dl at 1 h, 104 ,umole/dl at 2 h, and 107 diethyldithiocarbamate was omitted. This reaction is based on the ,umole/dl at 3 h. and the child became drowsy with mild ataxia release of [3HzO] from L-tyrosine (side-chain-2, 3, _3H; Amer which lasted 4 h. An open liver biopsy, performed at 25 months sham) the [3HzO] being distilled in Thunberg tubes and measured (after written informed consent was obtained) was histologically by liquid scintillation counting. 4HPPD was measured by method normal. II of Lindblad et at. (17). The incubation mixture contained Further clinical course. On day 24 the infant was dismissed in bovine liver catalase (Sigma, 0.8 g/liter), glutathione (10 mM), 2, good condition, on a low-protein diet (1 g/kg/day). There was no 6-dichloroindophenol (0.15 mM), Tris buffer, pH 7.5 (0.2 M) and recurrence ofsymptoms except for a painful erythema ofthe limbs 4-hydroxyphenyl-[1- 14CJ pyruvate [0.2 mM; 5.2 ,uCi/mmole, gen that disappeared after a few days. Serum tyrosine remained high erously provided by Dr. J. H. Fellman (7)] in a final volume of2.0 despite protein restriction. ml. The sample was pre-incubated for 20 min at 4°C with all the The girl has been on a regular diet since the age of 30 months above components except 4-hydroxyphenylpyruvate. This was and has continued excellent growth and psychomotor develop then added and the mixtures incubated at 37°C for 15 min in ment. When last seen, at 3% years of age, her protein intake was stoppered vials fitted with a filter paper disc containing 0.04 ml of normal and she was doing well. Her height was 96.5 em (25th hyamine hydroxide (New England Nuclear) suspended from the percentile) and her weight was 15.4 kg (50th percentile); physical stopper. The reaction was stopped with 0.2 ml of I N sulfuric acid examination was normal. The serum tyrosine level was 69.6 and incubation continued for a further 30 min to allow absorption 14 ,umole/dl (Table I). of the evolved [ COZ] on the filter paper. Radioactivity was Gas chromatography o/tyrosine metabolites. Urine was acidified determined by liquid scintillation counting in 10 ml of 0.4% to pH I, extracted 3 times with 2 volumes of ethyl acetate, dried omnifluor (New England Nuclear) in toluene:ethanoI4:1. Lactate over sodium sulfate, filtered, evaporated in vacuo and derivatized dehydrogenase (E.C. 1.1.1.27) and 6-phosphogluconate dehydro with a 1:1 mixture of Sil Prep (Applied Science) and BSTFA genase (E.C. 1.1.1.44) were determined as described previously (Pierce) at room temperature, overnight. Phenolic acids were (15) and glutamate dehydrogenase (E.C. 1.4.1.3) by the method of quantitated on a Hewlett-Packard model 5830 A gas chromato Beaufay et al. (I). Protein was determined by the method ofLowry graph equipped with a hydrogen flame ionization detector and an et at. (20). 8 ft x 2 mm column of 3% OV101 on 80/100 mesh Supelcoport Km values of tyrosine and a-ketoglutarate for TAT were deter (Supelco Inc.), using 4-phenylbutyric acid as internal standard. mined on undialysed cytosol fractions and calculated from Line Identity of compounds was confirmed by gas chromatography/ weaver-Burke double reciprocal plots. The Km for pyridoxal mass spectrometry on a Finnegan model 4021 mass spectrometer phosphate was obtained on samples which had been dialysed equipped with an all glass jet separator. Selective ion monitoring twice against 25 volumes of 0.2 M potassium phosphate, pH 7.4, at 302, 287, 212, 204, 195, 147, 123,95, and 81 m/e was used in an containing 5 mM mercaptoethanol and 1 mM EDTA, for 2 h each attempt to detect cis- or trans-4-hydroxycyclohexylacetic acids (4). Because dialysis did not result in complete removal of pyri (22). doxal phosphate, as evidenced by the appreciable enzyme activity Hepatic enzyme studies. Control liver was obtained within 4 h of in samples to which no pyridoxal phosphate was added, a correc death from children who died of trauma (#177 age 15 yr), lactic tion was made for the residual amount of pyridoxal phosphate in acidosis (#186, age 4 yr), nonketotichyperglycinemia (#240 age the sample after dialysis by extrapolating the curve of velocity 17 months), and Menkes disease (#262 age 28 months). Control versus substrate to zero velocity. The approximate Km for pyri 247 was a biopsy from a patient of 14 months with hypermethion doxal phosphate was then obtained from the double reciprocal inemia but no tyrosinemia or evidence of hepatic disease and plots. control 178 was a biopsy of normal hepatic tissue from a 3-year old patient with a hepatic hamartoma. Fresh tissue was homogen RESULTS ised in 19 volumes of 0.25 M sucrose containing 10 mM Tris or phosphate buffer pH 7.5, and centrifuged at 1000 X g for 10 min. The patient's urine gave a positive reaction to 2,4-dinitrophen The supernatant was centrifuged at 20,000 X g for 30 min and the ylhydrazine, suggesting the presence of an a-ketoacid, and to resulting supernatant carefully separated from the mitochondrial nitrosonaphthol, indicating the presence of tyrosine metabolites. pellet. All procedures were carried out at 0-4°C. Fractionation of Serum and urine levels of tyrosine were much increased: 62 the patient's biopsy material was performed immediately as de ,umole/dl and 9.39 ,umole/kg/24 h, respectively. Other aminoacids scribed above. The fractions were maintained at -70°C for 3 wk, showed no consistent abnormalities. Serum methionine was nor shipped on dry ice from Italy to the USA where they arrived still mal except on one occasion when several other amino acids were frozen, and maintained at -70°C until assay. The mitochondrial also mildly elevated (Table 1). Urinary o-aminolevulinic acid was pellet was suspended in a small volume of 10 mM Tris, pH 7.5 normal. The urine contained high levels ofp-hydroxyphenyllactic and sonicated three times for 15 sec each at 30-40 watts on a acid (pHPLA, 3.80 mg/mg creatinine), p-hydroxyphenylpyruvic Braunsonic model 1510 sonicator with a micro probe. For meas acid (pHPPA, 0.80 mg/mg creatinine) andp-hydroxyphenylacetic urement of enzyme activity in unfractionated homogenate, tissue acid(pHPAA, 0.27 mg/mg creatinine). Only pHPAA (0.005-0.042 was homogenized in 10 mM Tris pH 7.5 as above, centrifuged at mg/mg creatinine) is normally detected in control urines (2). The CHRONIC TYROSINEMIA 27 identity ofthese metabolites was confirmed by mass spectrometry; no inhibitor, either of TAT or of 4HPPD, was present in the selective ion monitoring gave no evidence for the presence of cis cytosol. TAT activity was normal in whole tissue, slightly lower or trans-4-hydroxycyclohexylacetic acids. than normal in cytosol and approximately twice normal in mito The stability of the hepatic enzymes from control tissue to chondria. Mitochondrial TAT in tissues from three controls rep storage at -70°C was investigated (Fig. I). All showed slow resented 11-17% of the total TAT activity whereas in the patient decline but retained at least 50% of their original activity even it was 19% of the total; however, the activity of glutamate dehy after a 12-month period. drogenase, a mitochondrial enzyme, was much higher in the The results of the enzyme analysis of the patient's liver biopsy patient's cytosol than in the controls, suggesting considerable are shown in Table 2. 4HPPD activity was undetectable in both rupture of mitochondria during the fractionation procedure. In whole homogenate and cytosol. Mixing experiments indicated that order to investigate whether the kinetics of TAT were normal, Km values for both substrates and coenzyme were determined (Table 3). o

• DISCUSSION e____ • Cytosolic TAT, the first and rate limiting enzyme of tyrosine metabolism, catalyses the transamination of tyrosine and a-keto 50 CYTOSOL glutarate to pHPPA and glutamate and requires pyridoxal phos e-e 4-hydroxyphenylpyruvote dioxygenose phate as coenzyme. In the mitochondria, tyrosine can be transa 0-0 tyrosine ominotronsferose minated by aspartate aminotransferase (EC 2.6.1.1), which utilizes _. 6-phosphogluconote dehydrogenose a wide range of substrates (21). It is not thought to be involved in 0-0 loctate dehydrogenase ..- .. glutamate dehydrogenase normal tyrosine catabolism. The second enzyme in the pathway, ___------..1.-. 1 --l...-..:..-_.. __l----.J 4HPPD, converts pHPPA to homogentisic acid. Absent activity of hepatic cytosol TAT (tyrosinemia type II) is associated with the Richner-Hanhart syndrome, tyrosinemia, and tyrosyluria. The eye .. and skin lesions respond rapidly to dietary tyrosine restriction. The enzyme defect was first described by Fellman et al. (9) in the ...... patient subsequently reported by Kennaway and Buist (13); mi . 50 ..... tochondrial TAT and 4HPPD were normal. In this condition pHPPA, the product of the missing enzyme is thought to be formed by mitchondrial transamination oftyrosine in many tissues which lack the 4HPPD. The pHPPA thus formed would enter the blood stream and utimately be cleared by the kidney (6, 14). . Table 3. Kinetic characteristics ofcytosol tyrosine aminotransferase 50 Control Patient WHOLE HOMOGENATE .. 0 V max' 0.88 0.37 Km for tyrosine 4.0 X 10-3 M 4.5 x 10-3 M Km for a-ketoglutarate 75 X 10-6 M 98 X 10-6 M Km for pyridoxal phosphate2 4.0 X 10- 6 M 2.1 X 10-6 M

2 4 6 8 10 12 I (fLmole/mg protein/h). Months in Storage 2 These values are approximations (see "Materials and Methods" sec Fig. I. Stability of hepatic enzymes to storage at -70°C. tion).

Table 2. Enzyme activities in liver fractions l Tyrosine amino- 4-Hydroxyphenylpyruvate Protein Time in storage transferase dioxygenase LDH 2 6PGD2 GDH 2 (mg/g wet wt) Whole homogenate Control 177 18 months 0.13 0.43 24 1.1 2.2 172 Control 178 13 months 0.14 0.67 44 1.3 2.5 129 Control 186 II months 0.10 0.61 44 2.3 1.7 126 Control 240 13 wk 0.27 0.31 39 1.5 1.6 137 Control 262 12 days 0.30 0.36 28 1.6 1.6 152 Patient K.W. 14wk 0.23 undetectable 31 1.6 1.7 102 Cytosol Control 247 7 days 0.36 0.38 37 2.0 0.25 155 Control 240 13 wk 0.44 0.55 74 2.3 0.49 88 Control 262 12 days 0.27 0.64 55 1.7 0.34 97 Patient K.W. 12wk 0.23 undetectable 38 2.2 2.5 68 Mitochondria Control 247 7 days 0.09 1.5 78 Control 240 13 wk 0.12 1.9 37 Control 262 12 days 0.12 2.7 42 Patient K.W. 12 wk 0.24 3.1 15 , Units of measurement for tyrosine aminotransferase, 4-hydroxyphenylpyruvate dioxygenase, LDH, 6PGD, and GDH: fLmoles/mg protein/h. 2 LDH, lactate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; and GDH, glutamate dehydrogenase. 28 GIARDINI ET AL. Although many cases of tyrosinemia type II have been recog liver showed no evidence ofan inhibitor ofthis enzyme, suggesting nized, absent cytosol TAT activity has only been reported in one that 4HPPD deficiency represents the primary enzyme defect in other patient (16); however, Goldsmith et at. (11) described a this form of tyrosinemia. 4HPPD catalyses a complex reaction mildly retarded 55-year-old male with the Richner-Hanhart syn which involves hydroxylation of the aromatic ring, decarboxyla drome and tyrosinemia. Hepatic cytoplasmic TAT was half the tion, and rearrangement of the side chain. Because our assay mean control value and although within the control range, this method is based on the release of CO2 from pHPPA, the defect in was assumed to be responsible for the tyrosinemia. It seems to us our patient must be at an earlier step of this enzyme activity than unlikely that tyrosinemia would result from a reduction of only in Danks' patient who excreted hawkinsin and the hydroxycy 50% of normal enzyme activity. These findings could however be clohexylacetic acids, metabolites presumed to be formed after explained by a mutant TAT with decreased affrnity for substrate hydroxylation and decarboxylation of pHPPA. This is consistent or cofactor, because the enzyme assays were presumably per with our failure to demonstrate these metabolites in the urine formed under saturating concentrations. Alternatively, this patient from our patient. Absence ofmetabolic acidosis in our patient and might represent a case of 4HPPD deficiency. Unfortunately, this most other patients with tyrosinemia suggests that the more com enzyme activity was not measured. monly observed tyrosine metabolites, pHPPA, pHPLA and Tyrosinemia and tyrosyluria were reported in another patient, pHPAA, are cleared more rapidly by the kidneys than the acids aged 3 years, with developmental delay and seizures but no other which accumulate in Danks' patient. manifestations of the Richner-Hanhart syndrome (4). Enzyme The specific activity of TAT in mitochondria from the patient assays in cultured skin fibroblasts revealed normal cytosol TAT was twice normal, a finding previously observed in two patients under saturating conditions, normal Vmax and Km for tyrosine, with cytosol TAT deficiency (10, 15). Although TAT activity in but markedly decreased Km for pyridoxal phosphate. 4HPPD was cytosol was close to the lower limit ofnormal, the apparent rupture normal. This report is interesting, not only because of the unique ofa major proportion ofmitochondria suggests that a considerable nature of the enzyme defect but because enzymes were detectable amount of the cytosol TAT activity could have derived from this for the first time in cultured skin fibroblasts. This conflicts with source; thus, it is not possible to rule out at least a partial deficiency our failure to detect these enzymes in fibroblasts but this could be ofcytosol TAT in this patient. It does however seem unlikely that due to differences in the culture conditions or some other unknown this represents the primary enzyme defect, first because it is factor. These reports illustrate the importance ofstudying enzyme certainly not totally deficient and second because the kinetics of kinetics in patients with apparently normal levels of enzyme the enzyme in the patient were entirely normal. activity. In conclusion, we have described a patient with transient ataxia Several other patients with tyrosinemia and tyrosyluria have and drowsiness and no persistent clinical abnormality, who has been described who do not clearly fit the picture of tyrosinemia tyrosinemia and tyrosyluria associated with deficient activity of type I or II. The patient described by Louis et al. (19) was a 35 hepatic 4HPPD. It is possible that several other patients, particu year-old male with only moderate mental retardation and marked larly those described by Wadman et ai. (25), Faull et ai. (5), and tremor of the hands, head, and feet. Enzymatic studies of this Goldsmith et ai. (11) may have had a similar enzyme defect. patient were inconclusive but suggested a possible deficiency of 4HPPD activity (5). Wadman et ai. (25) described a patient with REFERENCES AND NOTES severe mental retardation and cataracts; enzyme studies were not I. Beaufay, H., Bendall, D. S., Baudhuin, P., and deDuve, C: Tissue fractionation performed. Finally, Danks et ai. (3, 22) reported a patient with studies. 12 Intracellular distribution of some dehydrogenases, alkaline deoxy mild prolonged, transient tyrosinemia who presented with severe ribonuclease and iron in rat-liver tissue. Biochem. J., 73: 623 (1959). metabolic acidosis in whom the unusual tyrosine metabolites, 2. Crawhall, J. C, Marner, 0., Tjoa, S., and Claveau, J. C: Urinary phenolic acids hawkinsin and cis- and trans-4-hydroxycyclohexylacetic acid were in tyrosinemia. Identification and quantitation by gas chromatography-mass spectrometry. Clin. Chim. Acta, 34: 47 (1971). found in the urine. It was postulated that these metabolites were 3. Danks, D. M., Tippett, P., and Rogers, J.: A new form of prolonged transient derived from an intermediate of the 4HPPD reaction and that the tyrosinemia presenting with severe metabolic acidosis. Acta Paediatr. Scand., child and her mother were heterozygous for a defect of this 64: 209 (1975). enzyme which was able to oxidize and decarboxylate pHPPA but 4. DeGroot, G. W., Dakshinamurti, K., Allan, L., and Haworth, J. c.: Defect in soluble tyrosine aminotransferase in skin fibroblasts of a patient with tyrosi was unable to rearrange the intermediate to homogentisic acid. A nemia. Pediatr. Res., 14: 896 (1980). second family, with five affected members in three successive 5. Faull, K. F., Gan, I., Halpern, B., Hammond, J., 1m, S., Cotton, R. G. H., and generations, has recently been described (26). Danks, D. M.: Metabolic studies on two patients with nonhepatic tyrosinemia The patient described here differs from all other reported using deuterated tyrosine loads. Pediatr. Res., 11: 631 (1977). 6. Fellman, J. H., Buist, N. R. M., Kennaway, N. G., and Swanson, R. E.: The patients with prolonged tyrosinemia because ofthe absence ofany source ofaromatic ketoacids in tyrosinaemia and phenylketonuria. Clin. Chim. persistent clinical symptom. In particular, her development is Acta, 39: 243 (1972). normal, she does not have liver or kidney dysfunction, nor does 7. Fellman, J. H., Fujita, T. S., and Roth, E. S.: Assay, properties and tissue she have the eye or skin lesions characteristic of tyrosine toxicity distribution ofp-hydroxyphenylpyruvate hydroxylase. Biochim. Biophys. Acta, 284: 90 (1972). in the Richner-Hanhart syndrome. The cause of her transient 8. Fellman, J. H. and Roth, E. S.: Inhibition of tyrosine aminotransferase activity ataxia and drowsiness is unknown although, on at least one by 1.-3, 4-dihydroxyphenylalanine. Biochemistry, 10: 408 (1971). occasion, it was associated with an increase in the concentration 9. Fellman, J. H., Vanbellinghen, P. J., Jones, R. T., and Koler, R. D.: Soluble and of serum tyrosine. The child's normal psychomotor development mitochondrial forms of tyrosine aminotransferase. Relationship to human tyrosiriemia. Biochemistry, 8: 615 (1969). (she learned very quickly to speak Italian) indicated that the 10. Garibaldi, L. R., Siliato, F., de Martini, I., Scarsi, M. R., and Romano, C: hypertyrosinemia per se cannot account for mental impairment. Oculocutaneous tyrosinosis. Helv. Paediat. Acta, 32: 173 (1977). This has previously been suggested by Pelet et ai. (23) with II. Goldsmith, L. A., Thorpe, J., and Roe, CR.: Hepatic enzymes of tyrosine reference to four other patients (5, 10, 12). metabolism in tyrosinemia II. J.lnvest. Dermatol., 73: 530 (1979). Our patient has persistent hypertyrosinemia and tyrosyluria. 12. Halvorsen, S. and SkjelkvAle, L.: Tyrosine aminotransferase deficiency (TATD); first case diagnosed on newborn screening and successfully treated with Phe The serum tyrosine was not modified by high doses ofvitamin C Tyr restricted diet from early age. Pediatr. Res., 11: 1017 (1977). nor, surprisingly, by a low protein diet. Strict adherence to this 13. Kennaway, N. G. and Buist, N. R. M.: Metabolic studies in a patient with hepatic diet could not be verified and might explain this finding; in view cytosol tyrosine aminotransferase deficiency. Pediatr. Res., 5: 287 (1971). of the mild clinical course, more severe tyrosine restriction was 14. Kennaway, N. G., Buist, N. R. M., and Fellman, J. H.: The origin of urinary p hydroxyphenylpyruvate in a patient with hepatic cytosol tyrosine aminotrans not felt warranted. The absence of activity of 4HPPD in liver is ferase deficiency. Clin. Chim. Acta, 41: 157 (1972). unlikely to be due to instability of this enzyme to freezing for 12 15. Kennaway, N. G., Harwood, P. J., Ramberg, D. A., Koler, R. D., and Buist, N. wk because enzymes in the control tissue showed little decline in R. M.: Citrullinemia: enzymatic evidence for genetic heterogeneity. Pediatr. activity over this period, whether stored as intact tissue or super Res., 9: 554 (1975). 16. Lemonnier, R., Charpentier, C., Odievre, M., Larregue, M., and Lemonnier, A.: natant fraction. In all controls studied, 4HPPD activity was readily Tyrosine aminotransferase isoenzyme deficiency. J. Pediatr., 94: 931 (1979). detectable even in tissues stored up to 18 months. The patient's 17. Lindblad, B., Lindstedt, G., Lindstedt, S., and Rundgren, M.: Purification and CHRONIC TYROSINEMIA 29

some properties of human 4-hydroxyphenylpyruvate dioxygenase (I). J. Bioi. 24. Soupart, P.: Free amino acids of blood and urine in the human. In Holden, J. T. Chern., 252: 5073 (1977). (Ed.) Amino acid pools. p. 220 (Elsevier, Amsterdam 1962). 18. Lindblad, B., Lindstedt, S., and Steen, G.: On the enzyme defects in hereditary 25. Wadman, S. K., Van Sprang, F. J., Maas, J. W., and Ketting, D.: An exceptional tyrosinemia. Proc. Nati. Acad. Sci. (USA), 74: 4641 (1977). case of tyrosinosis. J. Ment. Defic. Res., 12: 269 (1968). 19. Louis, W. J., Pitt, D. D., and Davies, H.: Biochemical studies on a patient with 26. Wilcken, B., Hammond, J. W., Howard, N., Bohane, T., Hocart, c., and Halpern, "tyrosinosis." Australia N. Zealand J. Med., 4: 281 (1974). B.: Hawkinsinuria. A dominantly inherited defect oftyrosine metabolism with 20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein severe effects in infancy. N. Engi. J. Med., 305: 865 (1981). measurement with the Folin phenol reagent. J. Bioi. Chern., 193: 265 (1951). 27. We are grateful to Christine Jensen for performing the gas chromatographic and 21. Miller, G. E. and Litwack, G.: Purification, properties and identity of liver mass spectrometric analysis and to Drs. N. R. M. Buist and J. H. Fellman for mitochondrial tyrosine aminotransferase. J. Bioi. Chern., 246: 3234 (1971). their helpful discussion and advice. 22. Niederwieser, A., Wadman, S. K., and Danks, D. M.: Excretion ofcis- and trans 28. Requests for reprints should be addressed to: Dr. Nancy G. Kennaway, Dept. of 4-hydroxycyclohexylacetic acid in addition to hawkinsin in a family with a Medical Genetics, Oregon Health Sciences University, 3181 S.W. Sam Jackson postulated defect of4-hydroxyphenylpyruvate dioxygenase. Clin. Chim. Acta, Park Rd., Portland, OR 97201 USA. 90: 195 (1978). 29. This work was supported by a Biomedical Research Support Grant from the 23. Pelet, B., Antener, I., Faggioni, R., Spahr, A., and Gautier, E.: Tyrosinemia Oregon Health Sciences University. without liver or renal damage with plantar and palmar keratosis and keratitis 30. Received for publication October 21, 1981. (Hypertyrosinemia type II). Helv. Paediat. Acta, 34: 177 (1979). 31. Accepted for publication May 9, 1982.